THE EFFICACY OF RUELENE AS AN ANTHELMINTIC IN BEEF CATTLE

by

DANIEL ALBERT OSTLIND

B. S., Bethany College, 1958

A MASTER'S THESIS

submitted In partial fulfillment of the

requirements for the degree

MASTER OF SCIENCE

Parasitology

Department of Zoology

KANSAS STATE UNIVERSITY Manhattan, Kansas

1962 LP

*£ X. TABLE OF CONTENTS OotUWOyJTS

INTRODUCTION 1

LITERATURE REVIEW I

MATERIALS AND METHOOS 12

Laboratory Diagnostic Techniques 12

Anthelmintics 15

Statistical Procedures 16

EXPERIMENTAL RESULTS 17

Anthelmintic Studies 17

Larval Identification 40

DISCUSSION H

SUMMARY 53

ACKNOWLEDGMENTS 56

LITERATURE CITED 57 INTRODUCTION

Because of the economic Importance of the beef cattle Industry In the

United States and throughout the world, the losses Incurred among cattle by

gastrointestinal parasites are Important. There Is a need for practical and

efficient ways to combat Internal parasites. Therefore, the development of

safe and effective anthelmintics is necessary to supplement management practices.

However, before any parasiticide is placed on the market or made available to

veterinarians, it must be thoroughly tested. All aspects of the compound must

be investigated, including toxicity of the chemical and its metabolites, elim-

ination from host and residue, and of course, the anthelmintic activity at

prophylactic and therapeutic dosages.

The object of this study was to evaluate the effects of a new phospho-

organic systemic parasiticide, administered by different methods at varying

dosages, against naturally acquired nematodiasls in beef cattle.

LITERATURE REVIEW

Since the discovery of the systemic Insectictda) properties of organic

phosphorus compounds by the Germans, investigations have been conducted con-

cerning the effectiveness of such materials as anthelmintics as well as

insecticides.

In 1956 Trolene* (Dow ET-57, Dow ET-14, Korlan, Ronnel, 0,0-dlmethyl,

0-2,4, 5-trlchlorophenyl phosphorothioate) was marketed as a systemically active

drug against migrating stages of warbles in cattle. Since the work of Llndquist

(1956) many Investigators have shown the drug's efficiency against Hypoderma

*Dow Chemical Company, Midland, Michigan. a a

lineatum (DeVill.) and H. bovis (Linn.). However, as an anthelmintic Trolene

does not match Its grublcldal qualities. Crenshaw (1956) reported that

Trolene at the rates of 125 and 150 mg/kg of body weight would control

Haemonchus , Ostertagia and Cooper! In cattle. However, his results were

based on treatment of only two yearling calves. Worley (1957) noted that a

single dose at the rate of 110 mg/kg of body weight decreased the average EPG

(eggs per gram of feces) from 19. k to 3.2 In wintering Hereford cattle.

Herllch and Johnson (1957) performed critical tests on 8 naturally

Infected grade Jersey steers treated at the rate of 100 mg/kg. They found

that Trolene removed (species followed by per cent) Haemonchus place! 98,

Cooper i a punctata 59, Ostertagia ostertag! 46, Oesophagostomum radiatum 30,

Trichurls spp. 9 and Trichostrongylus axel 0.4. It was not effective against

T. colubriformis or Nematodirus hel vet i anus . Wood (1958) found Trolene In-

effective against experimental Infections of T. colubriformis in rabbits. Rlek

(1958) found Trolene to be effective only against H. place! and Cooper i spp.

at the rate of 5 g/100 lbs. In calves. He also reported an erratic control of

0. radiatum . Rlek and Keith (1959) found the same results In similar tests also

Involving calves. Allcata (I960), using critical tests, administered Ronnel

In bolus form (5 g/100 lbs) to 6 calves and found It to have an average efficacy

of 79.7 per cent. The calves had been experimentally infected with Cooper I a punctata . Drudge et al . (1961) treated beef and dairy cattle with a Ronnel bolus (100 mg/kg) and as a 10 per cent premlx at 2 levels (total dose of each,

105 mg/kg) and as a 0.75 per cent spray. Using pre- and post- treatment EPG counts to evaluate the drug's anthelmintic activity, he found that the bolus and premlx produced marked EPG reductions while the spray had no effect.

Schad et al. (1958) performed critical tests on range ewes that had been drenched with Trolene at the rate of 100 mg/kg. They found the drug to be

Ineffective against Haemonchus . Ostertagia , Tr i chost rongy 1 us . Nematodirus ,

Chabertia and the tapeworm Thysanosoma . Similar results were reported by

Gordon (1953a) In sheep treated at the rate of 5 g/100 lbs. At twice the dose,

he noted that it would remove H. contortus . Based on fecal egg counts, Dorney and Todd (1959) found Trolene, given orally to lambs at rates of 200, 400, and

600 mg/kg, effective against Haemonchus , Ostertagia , Strongyloides , and

Nematodirus . Trolene, given In a gelatine capsule at a rate of 100 mg/kg was not effective against an experimental infection of T. axei in 2-month-old lambs (Gibson, I960).

Levlne et al. (1956, 1958a), using horse strongyle larvae, studied the larvicidal activity of several organic phosphorus compounds including Chlorthion,

Halathlon, Diazlnon, 00VP, OS 1808, CoRal, Dipterex and Trolene. Trolene was the only compound inactive at 0.1 Molar (2.5 per cent) concentration. CoRal and Dipterex were larvicidal at 0.0074 M and 0.0001 M concentrations, respec- tively. However, Kelley and Marsh (I960) found both Trolene and CoRal to be ineffective against migrating larvae of Ascaris suum in baby pigs.

Levlne, Kantor and Taylor (1958b) determined the relative toxicities of some of the mentioned compounds In sheep and mice. In mice the LDcq's for DfclP and OS 1808 were fairly low, being 30 and 15 mg/kg, respectively. CoRal at

100-200, Dipterex at 600-800 mg/kg were less toxic, followed by the least toxic of all, Chlorthion at 1000-1200 mg/kg. Two of the k nematocides they tested In sheep, OS 1808 and 0,0,0-trl-r.-propyl phosphorotrlthlolte, had no effect on the parasites but were toxic to the host. Dipterex given to sheep produced toxic symptoms at a 400 mg/kg dose, however, the animals recovered*

Because of the limited amount of DDVP available their results were incomplete a a

concerning the effective and toxic closes. In limited laboratory tests Riek and Keith (1959) found Chlorthion, Malathion, Dtaztnon and DOVP ineffective as anthelmintics.

CoRal* (Bayer 21/199, Asuntol, Muscatox, 0,0-diethyl 0-(3-chloro-4 methyl -7-coumarinyl) phosphorothioate), an organic phosphorus insecticide developed by Bayer of Germany and marketed by Chemagro, was approved for use as a grublcide in cattle soon after the appearance of Trolene. As a result of critical tests in cattle and sheep, Herlich and Porter (1958) stated that a

CoRal drench at the rate of 25 mg/kg was highly effective ajainst Haemonchus .

Ostertagia . Trlchostrongylus , Cooper i . Trichuris . Capil larla . and Strongyloides

papil losus . At this rate toxic effects were mild and transitory. They also found an erratic control of Nematodirus spp. and Bunostomum phlebotomum . Except for the lack of activity against T. axei and £. phlebotomum . Rlek (1958) had similar results in cattle given a dose of 0.25 g/100 lbs. Gordon (1958a) found

the drug effective against H. contortus , Trichostrongylus spp. and JO. radiatum at the rate of 22 mg/kg but it also resulted In toxicosis and death to a few sheep. Although the drug was effective against parasites in the abomasuro and

small intestine of sheep, Gaivin et al . (1959) lost 13 of 15 animals treated at the rate of 25 mg/kg. In tests with calves he found good control of the

intestinal parasites in the same organs with only mild cases of toxicosis. In further tests with lambs and calves, Gaivin et al. (1960a) found the drug to

be very active against Haemonchus spp. and Cooper i spp. The dosages used were

3-12.5 and 12.5-50 mg/kg in lambs and calves, respectively. Only one lamb showed signs of toxicosis while two calves died at the higher dosages. Ounsmore

*Chemagro Corporation, Kansas City, Missouri. (I960) Injected Bayer 21/199 Into the rumen of sheep at 3 g/100 lbs. and found

It Ineffective against the abomasal parasites. Knight et al. (I960) reported

that a 15 mg/kg drench of CoRal was effective against H. contortus . Cooper I a

spp., T. colubriformls and Nematodirus spp. Eight of 90 sheep Involved died of

toxicosis. As a 0.25 per cent spray applied to cattle, the drug showed no

anthelmintic activity based upon EPG counts (Drudge et al., 1961). Baker

et al. (I960), Bailey and Walker (1961) used CoRal at the rate of 20 mg/kg to

treat gastrointestinal parasitism In cattle. In both cases the animals which

failed to respond to phenothlazlne were clinically Improved.

Bayer L13/59* (Dlpterex, Neguvon, chlorophos, 0,0-dlmethyl 2,2,2-trlchloro-

l-hydroxy ethyl phosphonate) , although better known as an insecticide, has

been tested as an anthelmintic by several workers. Gordon (1958a) treated sheep

with non-toxic doses and found It highly effective against H. contortus . When

taken directly into the abomasum it would also remove Trlchostrongylus spp.

Control of Oesophaqostomuro r ad latum was also observed but It was not dependable.

Rlek and Keith (1958) found Neguvon at the rate of 2 g/100 lbs. active against

_H. placel and 0. rad latum in cattle. At a higher dose (5 g/100 lbs.) It would

also remove Bunostomum phlebotomum . Cooper la spp. and T. axei . They observed

that better results were obtained against Cooperia spp. when the drug entereo directly Into the abomasum. In another experiment Riek (1958) found similar results. Allcata (I960) obtained good control of experimental £. punctata

Infections In calves by drenching with Bayer LI 3/59. The drug was safe at the recommended dosages. Dunsmore (i960) also reported that Dlpterex was more effective when introduced into the abomasum. Supperer and Pfelffer (i960)

*Chemagro Corporation, Kansas City, Missouri. treated calves with Neguvon at the rate of 90 mg/kg and found It highly

effective against H. con tortus with some activity against Ostertagia spp. In

sheep at the rate of 2.5 g/100 lbs. For treatment of acute 0. ostertagi in-

fections in young cattle. Banks and Mitton (I960) found that a dose of 69 mg/kg

was too low to be effective. However, dosages from 88-140 mg/kg produced

excellent results. Watt et al. (1961) used the drug at the rate of 80 mg/kg

and found that it reduced egg counts and made the animals more thrifty.

The results of Galvln, Bell and Turk (1959) showed that Bayer U3/59

(5 g/100 lbs.) was effective against abomasal but not intestinal parasites of

ruminants. In controlled tests against experimental Trichostrongylus axe?

Infections in 14-month-old sheep, the drug was ineffective as a 100 mg/kg

drench (Gibson, I960).

K'ung et al. (1959) administered Dipterex to sheep and goats by 2 sub-

cutaneous injections. The treatment was effective against Haemonchus . Bunos torninn ,

Nematodirus . Oesophaqostomum and Trichuris . However, because of the low worm

burdens, the results were suggestive rather than conclusive.

Dimethoate* (Compound CL 12,880, Rogor k0, 0,0-dimethyl S-(N-methylcar-

bamoylmethylphosphorodlthioate) has shown promise as a grublcide, although

reports on its possibilities as an anthelmintic are limited.

Drudge et al. (1961) fed dimethoate to cattle as a 10 per cent premix In

the grain ration at the rate of 3 mg/kg/day for 5 days. The treatment was

discontinued on the fourth day due to toxicosis. There were no significant

reductions in the egg counts made at the termination of the test.

Baker et al. (1959) treated Hereford heifers with Oowco 105** as a drench

at the rate of 120-130 mg/kg. The results showed that It removed 90-100 per

American Cyanamid Company, New York, New York. **Dow Chemical Company, Midland, Michigan. a

cent of Trichostrongylus spp., 87 per cent of Cooper 8 spp., and 66 per cent of Ostertagla spp. Five of the 10 animals used in the test showed toxicosis with one case being severe. Yearling feeder steers with acute parasitic in- fections which failed to respond to phenothiazine were drenched with a 100 mg/kg dose of Dowco 105 (Baker et al., i960). The drug proved very effective as judged by the clinical appearance of the animals. Douglas et al. (1959a) tested the anthelmintic activity of the same drug in sheep. The efficacy of the drug against all gastrointestinal parasites in the abomasum and small intestine was 93 per cent at a dosage of 200 mg/kg. When the dosage was reduced to 75 mg/kg the efficacy decreased to 55 per cent. No animals at either dosage showed signs of toxicosis.

Worley (1958) fed yearling feedlot cattle Dowco 109* at rates of \k mg/kg over 6 and 12 day periods and 15 mg/kg for one day. The drug showed no effect on the intestinal parasites based on egg counts. Dowco 109 differs from Dowco

105 by possessing a methyl rather than an ethyl group attached to the nitrogen atom (Plate 1). Ruelene** Is Identical to the structure of Dowco 109 except that an oxygen atom replaces a sulfur atom.

Four-tert*butyl-2-chlorophenyl methyl methylphosphoramldate (Ruelene,

Dowco 132) has shown promise as an anthelmintic with a broad spectrum of activ- ity and a wide margin of safety.

Sewell (1959), Ross and Karr (1959a) compared Ruelene with phenothiazine

for worming lambs on pasture. Ruelene at a 1 50 mg/kg dose reduced EPG counts significantly lower and the lambs gained better than those treated with phenothiazine. The Ruelene treated animals were also more thrifty. In dry

*Dow Chemical Company, Midland, Michigan. **Dow Chemical Company, Midland, Michigan. EXPLANATION OF PLATE I.

The structural formulas of some organic phosphorus compounds that have been tested as anthelmintics. PLATE I CI OCH3 CI DCH3 CH3 CH 0—P< CH3-C- 0— P< CH 3 NHCH3 CT~ 0CH3 Trolene Ruelene

CI OCH3 CI 0CH3 C^3 S CH3/ P< CH3-C 0— P< CH3-6-( 0— CH3 CH3 \ NHC2H5 NHCH-

Dowco 105 Dowco 109

OC2 H5 CH3O. Otf CI 0=C )p-c-6-ci

1 1 H CI Cl-C. OC2 H5 CH3O Neguvon '3 CoRal

CH3 CH3O.

1 —0—c=cV 9 -S—C—C— NHCH3 CI CH3O CH3O DDVP Dimethoate

CH CH 3 N 3 N p§ 2 c—B C2H5 > —s—c— 0— — )N0 2 CH3O _ CH3O CI H_i_g_ C2H5 H

Chlorthion Malathion 10

lot experiments Ross and Karr (1959b) compared Ruelene and phenothfaztne

drenches for worming fattening lambs. The results were similar to the pasture

tests as Ruelene produced better gains and lower egg counts. Weight gains

of 2-year-old Texas ewes drenched with 2 cc'10 lbs. of either phenothlazlne

or Ruelene were compared by Ross and Brooks (I960). The Ruelene treated

ewes had better gains and showed a thriftier appearance.

In 3-month-old lambs treated with a 200 mg/kg Ruelene drench, Douglas and

Baker (1959b) found that Osterragla spp., Trtchostrongylus axe? . T. vitrinus

and Nematodirus spp. were effectively controlled. The drug was not as active

against T. colubriformls . At this rate the drug produced no signs of toxicity.

Shaver and Landram (1959) reported that Ruelene as a drench appeared to be

effective in sheep, goats, and cattle for the control of Haemonchus . Cooper la ,

Ostertaqia . Tr i chos t rongy 1 us . Oesophagostomum and Strongyloldes spp. They

suggested dosages of k0, 100 and 125 mg/kg for cattle, sheep, and goats, res-

pectively. At these rates no adverse reactions were observed. Mull i son and

Shaver (I960) treated cattle with Ruelene at the rate of kO mg/kg. Five days after treatment the egg counts were reduced by 89 per cent. Administration of the compound as a spray did not affect the EPG's. In sheep treated at the

rates of 200, 100 and 50 mg/kg, they found the egg counts reduced by 99, 9^ and 93 per cent, respectively.

Kenny (i960) used Ruelene to treat lambs suffering from acute parasitic gastro-enteritts. He drenched the lambs on 2 occasions at the rate of k g/100

lbs. The treated animals not only showed much improvement in appearance but

the weight gains were almost double that of the controls also. Post-mortem examination of the abomasums of some of the untreated sheep revealed gross numbers of Ostertagla circumcincta and Trlchostrongylus axei present in the s a

II

ratio of 12 to 1, respectively. Watt et al. (1961) treated bovine ostertaglo-

sis with Ruelene at the rate of 2 g/100 lbs. and found that egg counts were

reduced. The animals were also more thrifty. He observed only slight dis-

comfort In four animals due to toxic reactions from the drug.

Galvin, Bell and Turk (1960b) conducted 3 tests involving 30 lambs to

determine the anthelmintic and toxic effects of Ruelene. They concluded

that Ruelene (100 mg/kg) was not suitable for treatment of mixed infections.

They also stated that although higher doses (200 mg/kg) were more active

there were signs of toxicosis. Chili nesterase activity was reduced 50 per

cent by a 100 mg/kg dose and markedly depressed at higher dosages.

loset and Ludwlg (I960) reported on preliminary tests using Ruelene as a

"pour-on" for intestinal worm control. This method of application showed 89-

99 per cent efficiency based upon egg count reduction. Herllch et al. (1961),

using the topical application (75 mg/kg), reported erratic control of Cooper!

punctata . C,. oncophora and Oesophaqostomum radiatum . with no activity against

Ostertagla ostertagi or Trlchostrongylus axel . It was 100 per cent effective

against Haemonchus place! . In the same experiment they showed that Ruelene

administered orally at dose rates of kO to 60 mg/kg controlled £. place! .

£• punctata , and £. radiatum . At the highest rate It was also active against

Jp.. ostertagi . T. axel and T. col ubr? form! . However, at this rate It was

lethal to one of three steers.

Allcata (I960) drenched calves, that had been experimentally Infected

with C. punctata , with I.76 g/100 lbs. (39 mg/kg) of Ruelene. The results showed the anthelmintic activity to range from 98.9-100 per cent. No signs of toxicosis were observed in the 100 calves receiving the compound. Drudge et al. (1961) administered Ruelene at the rate of kk mg/kg as a drench to a

12

cattle tn 2 experiments. They found that the treatment reduced egg counts

86 per cent In dairy calves and 97 per cent in Hereford steers.

MATERIALS AND METHODS

Laboratory Diagnostic Techniques

Fecal egg counts were used to estimate the level of intestinal parasite

Infections In experimental animals. Egg counts were done according to the procedure of the modified Lane (1928) flotation technique of Dewhtrst and

Hansen (1961). With the use of this technique It Is possible to determine

the total egg count of each animal which is expressed as eggs per gram of feces

(EPG). This method also facilitates the separation of eggs into genera or groups of genera on the basis of morphological characteristics as determined with the aid of a compound microscope. Krug and Mayhew (19**9) showed the statistical reliability of the egg classification technique. Hansen and

Shivnani (1956) demonstrated the accuracy of the differential egg count method by culturing and identifying infective larvae. Using this method, the eggs

were placed in two groups and four genera. One group is the Cooper i .

Ostertagia and Trichostrongylus (COT), and the other group, Haemonchus and

Oesophagostomum (HO). The eggs of the four genera, Bunostomum , Nematodirus ,

Trichuris and Strongyloides are quite distinct and easily classified.

Strongyloides ova occurred sporadically and are primarily seen in feces of calves so they were not counted.

Although coccidian oocysts were present, no attempt was made to count them or record their presence.

Fecal samples used for making egg counts were obtained directly from the rectum or fresh fecal pads on the ground. The samples obtained from fecal 13

pads were either dropped by an animal under observation or collected at random

from the feedlot. Samples ranging from 5 to 25 grams were collected in small

glass jars, numbered and refrigerated at approximately 7°C until EPG counts

could be made. All egg counts were done within two weeks after collection

In order to reduce any variation In the count because of storage. Storage

results in changes in fecal consistency and morphological characteristics of

the eggs (Dewhirst, I960).

From each sample, 10 grams were weighed into a 300 ml. Erlenmyer flask on

a Harvard Trip Balance. Tap water was added to the 300 ml. mark. The entire

contents of the flask were poured into the standard 1000 ml. glass container

of a Waring Blendor. The samples were homogenized for 30 seconds in the

blendor. After homogenizing, approximately 50 ml. were strained into a beaker.

As quickly as possible after agitating the beaker, two centrifuge tubes were

filled to the 15 ml. mark* This step must be done quickly because the eggs will settle rapidly in water. The tubes were centrifuged for 3 minutes at

1000 r.p.m. in an Internationa] Model C50 centrifuge. After allowing the

centrifuge to coast to a stop, the supernatant was decanted, leaving the eggs

and solid matter packed at the bottom of the tube. The tubes were shaken by

hand to break up the packed mass before adding enough aqueous sodium nitrate

flotation solution (specific gravity 1. 35-1*^0) to fill the tubes and form a convex meniscus on top. An 18 mm* square No. 2 cover glass was carefully placed on the top of each tube and the tubes were centrifuged again for 3 minutes at 1000 cp.m. A Walser Automatic Timer, Model 8066-B, which automati- cally stopped the centrifuge, was employed to help standardize the technique.

After completion of the final centrifugation the two cover slips were care- fully removed and placed on a 2 x 3 Inch glass slide. The cover slips were \k

examined, using a standard procedure, under a compound microscope equipped with a mechanical stage. All counts were made using the lOx ocular and lOx objective (low power). The number and type of eggs were recorded on an Adams

Laboratory Counter.

For later experiments, the homogenizing and centrlfugatlon times were changed to five seconds and one minute, respectively. The change was Innovat-

ed because the use of the Waring B lender for over 10 seconds had been found to

reduce EPG counts as much as 50 per cent (Dewhirst, I960). The centrlfugatlon

time was reduced for convenience. In all cases, however, the procedure used

was the same for both pre-treatment and post-treatment fecal samples.

Larvae were cultured from thre to five grams of feces, spread by means

of a tongue depressor, on sterile gauze pads. The pads were placed In eight-

ounce wide-mouth bottles and moistened with two ml. of water. Lids were

placed on the jars loosely to allow entry of air. After Incubation at room

temperature (75-80°F.), for 8 to 10 days, each gauze pad was Baermannlzed,

fecal side up, 12 to 18 hours In warm water (85-90°F.). The larvae were

collected In shell vials which were corked, numbered and packed In dry Ice

for shipment to Manhattan, Kansas, from Fort Worth, Texas.

For critical tests, the digestive tract from the abomasum to the anus

was obtained from several animals during processing at a packing plant. Each

organ (abomasum, small Intestine and large Intestine) was separated and cut

open. The contents of each organ were flushed into a clean 10 gallon garbage

can. The gut lining was then scrubbed five or more times by hand. The total

washings and organ contents amounted to about five gallons per organ per

animal. The contents of each can were allowed to settle ^5 to 60 minutes

then each can was carefully decanted and refilled with tap water. The 15

settling, decanting and refilling process was repeated five or six times on the contents of each can. This process disrupts the solid matter and faclll* tates the concentration of the worms. The final concentration was poured

into one-half gallon jars and formal inized. The jars, one for each organ of each animal, were sealed and shipped to Manhattan, Kansas, from the Texas

Phenothlazine Company Lab. at Fort Worth, Texas.

The entire contents of each Jar were examined under a dissecting scope.

A petri dish was marked into sections with a red grease pencil so that each section occupied approximately two-thirds of the low power field. Five to 20 ml. were examined at a time, depending upon the amount of solid matter. The worms were collected with a hooked teasing needle and placed in distilled water for four hours. Then they were placed In 35 per cent alcohol for an additional k hours before being transferred to a 70 per cent alcohol -5 per cent glycerol preservative. All worms were identified to genus and to species when possible.

Anthelmintics

In this study, different dosages of luelene* (U-tert-butyl-2-chlorophenyl methyl methylphosphoramidate) were administered by various methods to cattle.

In only one test, phenothiazine in combination with lead arsenate, was com- pared with Ruelene.

The Ruelene used for oral treatment (via feed) was in the form of a wettable powder containing 25 per cent active Ingredient. The amount of drug

needed to meet the proper dosage per animal per day was calculated and weighed out before mixing with the feed ration. In one experiment Ruelene was mixed with the morning ration as a high-level and low-level treatment, 10 and 3 mg/kg

*Dow Chemical Company, Midland, Michigan. ,

16

of body weight per day for 5 days, respectively. In a second test, 6.1 pounds

of Ruelene formulation M-1839 was mixed with the total 3500 pound ration.

The entire 3500 pounds were consumed In k0 hours, which resulted In uptake of

the drug at the approximate rate of 25 mg/kg per day or a total dosage of kS mg/kg per head. About 25 or ]/k of the animals Involved showed excessive

salivation 12 to \k hours after feeding began. One heifer also showed severe

respiratory distress. After the second day all animals, Including the heifer,

had recovered.

The anthelmintic effects of Ruslene as a drench were Investigated In tests

using dosages of 50 and 75 mg/kg. There were no signs of toxicity In animals

treated with the 50 mg/kg dose. H >wever, slight salivation, some stiffness

and depression resulted from the 75 mg/kg treatments.

Emulslflable Ruelene was administered as a "pour-on"* at the rates of

50 and 75 mg/kg. The term "pour-on" describes a new topical application of

systemic compounds. This method consists of pouring a measured amount of

the drug on a small area of the animal's back.

With the 50 mg/kg "pour-on" treatmen' no toxic syratoms were exhibited by

the animals. However, In one of the tests using the 75 mg/kg "pour-on" about

20 animals showed slight salivation 2k hours after treatment while In other

tests the same dose produced no overt symptoms.

Statistical Procedures

Analyses of variance were used to measure the reliability of egg counts performed during the anthelmintic tests. Only for the Crofoot Feedlot exper-

iment was the egg count data transformed according to the formula >Jx-1

*Dow Chemical Company, Midland, Michigan. 17

x equals the particular egg count.

A test of repeatability performed on the larval sampling technique

revealed that It was reliable.

Ch! -square was used to test the validity of differences between EPG and worms post-mortem (WPM) ratios of control and treated animals. Chl-square was also used to measure differences between the number of each species of

larvae, before and after treatment, with respect to Ruelene, phenothlazlne

and the controls.

EXPERIMENTAL RESULTS

Anthelmln.ic Studies

Cleburne. Texas Feedlot Test (Tables 1-11, Plate II, III). Heifer and

steer calves of mixed breeds belonging to the Texas Meat and Provision Company,

Dallas, Texas, were used In this experiment. The animals, weighing about

feedlot 300 lbs., were purchased at auctions in central Texas and placed In a

near Cleburne, Texas. Many were treated for shipping fever or pneumonia

shortly after arrival. Most of the calves had been in the feedlot from two

to four weeks prior to testing.

The cattle were fed from bunkers having a capacity of six tons. The

feed was mixed by a local feed company and consisted of ground corn, 29.5;

cracked ml!©, 29.5; cotton seed meal, 13.5; cotton seed bulk, 20.2; ground

alfalfa, 5.3; salt and mineral supplement, 2 per cent, and 2 lbs. of Aurofac

10*/ton of mixed feed.

Ruelene was administered topically at 2 rates (50 and 75 mg/kg) and as

*Aureomycin Feed Supplement of American Cyanamld Company. New York, New York. 18

a 50 mg/kg drench.

Two hundred calves were divided Into two pens for the experiment. In

the first pen 50 head were tagged In the left ear and treated with Ruelene formulation M-1609 diluted with 2 parts of water. Using a calibrated dipper, about 3.5 oz. of the mixture were applied as a 50 mg/kg pour-on. The remaining

50 head were tagged In the right ear and treated with the same formulation, using about 5 1/3 oz. to provide a dose of 75 mg/kg. In the second pen 50 head were tagged In the left ear and given 3A oz. of Ruelene formulation

M-1782 using a standard drenching gun. The remaining 50 head were untreated controls. From the treated groups and controls, 12 rectal fecal samples were collected before treatment and again 13 days later from the same animals.

Results of the study based on EPG counts are given in Tables 1-3. Although

the treatments reduced the post-treatment egg counts, statistical analyses showed them to be non-significant (Tables 1-2). Grouping the data on the basis of pre-treatment egg counts showed the post-treatment EPG reduction of the treated animals to be highly significant (Table 3).

The animals were slaughtered after they had been fattened. The abomasum

(stomach), small and large intestines were recovered from animals 15^, 203,

207, 209, 258 (control) and 263 (control). All nematodes present in these organs were collected and identified (Tables *t-9).

The number of worms from the digestive tract of control animals was compared with the number recovered from treated animals that had either similar pre- or post-treatment egg counts (Tables 10 and 11). The worms recovered from treated animal. 203 (50 mg/kg drench) were compared with control 263, both of which had similar pre-treatment egg counts (Table 10, Plate II).

Control 263 had more than 5 times the number of worms present in 203. 19

and Cooper la were more Comparing the organs separately showed that Haeroonchus

Larval Identification susceptible to treatment than other nematodes present.

showed that per cent of the performed on a pre-treatment fecal sample of 203 15

resulted In 100 per cent eggs present were Haeroonchus . Therefore, treatment

of the 453 worms present control of Haeroonchus . Cooper la spp. represented 433

remaining 20 were composed of tn the small Intestine of animal 263. The organ of animal 203. several genera. Only 35 worms were present In the same

the remainder were of several other genera. Twenty-four were Cooper I a spp. while

Cooper la present. The worms Thus, the major reduction was In the number of with control 258 be- recovered from treated animals 15** and 207 were compared

had a similar pre- cause 154 had a similar post-treatment egg count and 207

that the abomasum from treatment count (Table 11, Plate III). It must be noted

because of the difference In the 154 and 258 cannot be compared with each other

small Intestine nematode populations. However, the nematodes present In the

is justified (Tables are similar as to species and number so that a comparison

from 702 to 116, It Is 4 and 8). Because the egg count of 154 was reduced portion of the reasonable to assume that the number of WPM represents only a approximately worms present before treatment. The post-treatment count Is

reduction Is changed one-sixth of the pre-treatment egg count. However, this

approximately 50 per cent. to 1/3 because the egg count of control 258 dropped

treatment if each egg Thus, there were 3 times as many worms present before ratio of the COT represented one worm. This is not the case, as the egg-worm over 40 times group was 1:13.57 (Oewhlrst and Hansen, 1961). Therefore,

prior to treatment. (3x13.57) as many WPM were present In the small Intestine conducted on animals This should be a conservative estimate as this study was

animals on pasture. In feedlot, whereas, Oewhlrst «s study (1961) involved 1

20

Table 1. The effect of dermal and oral treatments with Ruelene on egg counts In the feces of Texas cattle.

'. No No. ! Averaqe EPG Count S- Ranqe Per Cent Treatment j Smpld. ! 9/13/60 . 9/26/60 Reduction ^ j J

50 mg/kg 50 12 216(13-900) 34(4-113) -84.3 pour-on

75 mg/kg 50 12 285(37-709) 75(0-202) -73.7 pour-on

50 mg/kg 50 12 177(18-1014) 74.5(1-282) -58.0 drench

Controls 50 12 146(19-440) 117(13-372) -19.9

Table 2. Analyses of differences between pre-treatment and post-treatment EPG counts, Cleburne feedlot calves.

* • * Source of Variation d.f. : Ms : F : : :

Treatments 3 56273.16 1.22 ns Animals:Trts. 41 46293.67 ns - non-significant

Table 3. Analyses of variance of the grouped EPG data, Cleburne Feedlot Test.

Source of Variation ! d.f. ! Ms F • • •

Treatments 3 56273.16 23.52***

Animals:Trts:Grps 29 2392. 1

*** p < .001 21

Table 4. Nematodes recovered from animal No. 154.

| Organ Species Hale • Female Total \ • • • •

Abomasum Haemonchus spp. 106 106 Ostertagiaoo. 97 97 Haemonchus placet 81 81 Ostertagia ostertagl 62 62 Cooper Jagg 28 28 Haemonchus contortus 17 17

T r I chos t rongy 1 usoo. 13 13 Cooper i a punctata H 11 Haemonchus Immature 5 S Ostertagia lyrata 2 2

Tr i chos t rongy 1 us axei i 2 Subtotals, 183 5 236 424

Small Cooper iaoo 688 688

Intestine Cooper i a punctata 438 438

Cooper i a spp. 38 38 Bunostomum trigonocephalum 3 4 7 Nematodirus spp. 6 1 7

Ostertagia ostertagl 1 1

Tr 1 chos t rongy 1 us 1 1 longispicularis

Tr J chost rongy 1 usgo. 1 I

Subtotals, 487 694 1181

Large Oesophagostomum radiatum 1 4 5 Intestine Trichuris Immature 2 2

Trichuris spp. 1 1

Subtotals, 1 2 5 8

Totals, 671 7 935 1613 22

Table 5. Nematodes recovered from animal No. 203.

Organ Species : Hale : Female Total • 1 | • • • 1

Abomasum Ostertagia oo. ko ko Ostertagla ostertagl 20 20

Ostertagia lyrata 1 I

Bunostomum tri gonocepha 1 urn I 1

Cooper i a oo. 1 1

Subtotals, 22 41 63

Small Cooper i a co \k \k Intestine Cooper! a punctata 9 9

Bunostomum t r gonocepha 1 urn 2 * 6 i Ostertagiaocj 2 2 Strongyloiaes paplllosus 2 2

Cooper! a spp. 1 1

Nematodirusgcj I 1

Subtotals, 12 23 35

Large Intestine

Subtotals,

Totals, 3*» 6k 98 t

23

Table 6. Nematodes recovered from animal No. 207.

• * • • Organ ! Species ! Hale .' • • Female Total * • •

Abomasum Ostertag!a$$ 284 284 Ostertagta ostertagl 163 163 Trlchostrongylus^ 32 32 Ostertagia lyrata 8 8 Trlchostrongylus axel 7 7 Ostertagia Immature 2 2 Bunostomum trigonocephalum 2 2

Cooper la punctata 1 1

Subtotals, TiT 2 316 499

Small Ostertagl aoo 20 20

Intestine Ostertag I a ostertag 10 10 Nematodlrus spp» 4 3 7 Bunostomum trigonocephalum 1 4 5 Trlchostrongylus 3 3 longispicularis Trlchostrongylusoo^ 2 2 Ostertagia lyrata 2 2

Cooper 1 a punctata 2 2 Cooper ia^o. 2 2

Subtotals, 22 31 53

Large Unidentified Larvae 5 5 Intestine Oesophagostomum radiatum 1 3 4

Trichuris spp. 1 1

Trichuris Immature 1 1 "*4 Subtotals, 1 6 11

Totals, 204 8 351 563 Table 7* Nematodes recovered from animal No. 209.

: • 1 Organ Species Male Female ! Total * • • •

Abomasum 0stertagla99. 257 257 Ostertagla ostertagl 210 210 Trlchostrongylus °9 161 161 Trichostrongylus axel 99 99 Ostertagla lyrata 13 13 Subtotals, 322 418 740

Small Cooperla punctata 5 5 Intestine Cooper! a oo 5 5

Cooper i a spp. 2 2 Ostertagla ostertagl 2 2 Ostertagla $9- 2 2 Trlchostrongylu$9$ 2 JL Subtotals, 8 17

Large Intestine*

Totals, 331 426 757

Sample destroyed in shipment. —

25

Table 8. Nematodes recovered from animal No. 258.

: :

Organ • Species • Male Female : Total • • • •

Abomasum Ostertagia^g \0k \0k

Os tertag i a ostertag i 8k 8k Trlchostrongylusgg ko ko Trichostrongylus axel 31 31 Cooperia go 22 22 Cooperia punctata 7 7 Ostertagia lyrata 3 3 Haemonchus place) 2 2 Haemonchus contortus 1 1 Haemonchus spp. 1 1

Subtotals, 126 169 295

Small Cooperia go 773 773 Intestine Cooperia punctata 388 388 Cooperia spp. llii \\k Bunostomum trtgonocephalum 1 1 2 Nematodlrus spp. 1 1 Ostertagia 1 1 gg Trichostrongylus 1 1 longispicularis

1 T r 1 chos t rongy 1 usgg 1

i Subtotals, 505 776 1281

Large Unidentified Larvae 2 2 Intestine Trichuris spp. — _ 1 _J.

Subtotals, 2 1 3

Totals, 631 2 SkS 1579 1 »

26

Table 9. Nematodes recovered from animal No. 263*

Organ Species : Hale Female : Total

Abomasum Ostertagla oo 18 18 Trlchostrongylus 03 11 11 Ostertagla ostertagi 9 9 Haemonchus placet 9 9 Haemonchus spp. 6 6 Trlchostrongylus axel 5 5 Haemonchus contortus k 4 Cooper! a po 2 2 Subtotals. 20 kk 6U

Small Cooperla og 225 225 Intestine Cooperla ^punctata 176 176 Cooperla spp. 32 Trlchostrongylus qq 15 15 Nematodlrus spp. 1 1 2 Ostertaglaoo 1 1

Haemonchus placet 1 1 Bunostomum trigonocephalum 1 JL Subtotals, 210 2^3 *»53

Large Trichuris spp* 3 3 Intestine Trichuris Immature 2 2

Oesophagostomum rad latum 1 1

Unidentified Larva 1 1

MMMi «• 1 » «—i

Subtotals, 1 3 3 7

Totals* 231 3 290 52** 27

Table 10. Comparison of the total WPH of a treated and a control animal with similar pre-treatment egg counts.

EPG *** WPM *** Total ; Animal ' Treatment : Pre . Post /-.bo. . Sni. Int. . Lg.lnt. : wpm • » *

203 50 mg/kg 29 2 63 35 98 drench

263 Control 30 82 64 455 7 524

P < .001 as determined by Chi-square.

Table 11. Comparison of the total WPH of a control and two treated animals with similar pre- or post-treatment egg counts.

n .w* *** EPG WPM | Total Animal : Treatment : Pre . Post Abo. Sin. Int. Lg.lnt. WPM : : 1 :

207 50 mg/kg 208 21 499 53 11 563 drench

15^ 75 mg/kg 702 116 424 1181 8 1613 drench

258 Control 241 130 295 1281 3 1579

*** P < .001 as determined by Chi-square. EXPLANATION OF PLATE II.

Comparison of the total worms recovered from each organ of a treated animal and control.

EXPLANATION OF PLATE III.

Comparison of the total worms recovered from each organ of a control and two treated animals. 31

PLATE III a a

32

Kelly (1955) showed that with an Increase tn roughage the number of worms per egg Increased.

Because of similar pre-treatment egg counts and worm burdens, the number of worms present In the abomasum and small Intestine of animals 207 and 258 were compared. Ruelene treatment resulted in 100 per cent control of Haemon- chus as larval counts revealed that 8 per cent of the eggs present before

treatment were of this genus. Again, as between 15** and 207, there was a

between great reduction In the number of Cooper i as compared with Ostertagia

animals 207 and 258. The vast majority of worms in the small intestine were

Cooper la spp., while in the same organ of the treated animal, 30 of 53 worms

were Ostertagia spp. (Tables 6 and 8). This shows that Cooper i spp. are more

susceptible to Ruelene than Ostertagia spp.

Crofoot Feedlot Test (Tables 12, 13, Plate IV). Cattle of mixed breed,

averaging approximately 800 lbs., from southeastern Oklahoma and northeastern

Texas were used In this test. The animals were in the feedlot approximately

four weeks before the test began. Some of the animals were treated for ship-

ping fever. The animals were divided and placed in three separate pens. The

animals in 2 of the pens received a high and low dosage of Ruelene at the

rates of 10 and 3 mg/ kg/head/day for 5 days, respectively. Ruelene, In the

form of a wettable powder, was weighed Into calculated amounts and mixed with

the ration each morning. The antmals In the third pen were controls.

The ration was fed from bunkers and consisted of 8 to 9 lbs. grain (50%

cracked corn and 50% cracked mllo), I 3/k lbs. ground alfalfa hay, 8 lbs.

progressed sorgo silage and 1 lb. of k\ per cent protein supplement. As feeding

the grain was increased to about 22 lbs.

Rectal fecal samples were taken from 20 head that were numbered in each 33

of the treated lots. Fecal samples from the control animals were taken from

20 fresh pads at random throughout the pen. Samples were taken prior to, 2

days after treatment and twice a month until the animals were slaughtered

(Table 12). Fecal samples were taken from the same animals, whenever possible,

at each sampling date. Whenever a particular animal could not be sampled, a

random sample was taken from the lot so that egg counts could be done on a

total of 20 samples from each group. Plate IV shows the curves of the total

EPG's at the sampling dates of the treated and control animals.

Analyses of variance performed on the data after transformation showed

the low level rate to be just as effective as the high level dosage. Both

treatments reduced the EP6 counts significantly lower than the controls

(Table 13).

Ruelene Versus Phenothlazine-Lcad Arsenate Drench Test (Tables 14-17).

Ruelene and phenothiazine were administered as drenches to a group of culled

calves from the animals used in the Cleburne Feedlot Test. These calves had

not been treated. Ruelene formulation M-1732 was given at the rate of 75

nig/ kg to 32 head that had been ear-tagged. Two to 3 ounces of a phenothlazine-

lead arsenate drench, containing 15 gm phenothiazine and 0.25 gm lead arsenate

per ounce, were given to the remaining 36 head. Each group was weighed sep-

arately and fecal samples were collected from 10 marked animals in each group.

The calves were group-weighed again 16 days later at which time post- treatment

fecal samples were collected.

The egg counts of the Ruelene treated calves were reduced 99.2 per cent

from the pre-treatment levels while the EPG counts of the phenothiazine

treated animals were reduced 61.8 per cent (Table \k). However, the Ruelene

treated animals did not gain as well as those receiving the phenothiazine 34

Table 12. EPG Counts of Crofoot Feedlot Test Cattle.

£• Range Per Cent No. No. ! Average EPG Count \

Treatment 1 • . , . 12 30 Reduction md ^^ 0/20/5g / /59 :

Ruelene 201 80(3-242) 5(0-21) -93.6 50 rog/kg in feed

Ruelene 267 20 50(6-407) 6(0-40) -88.0 15 mg/kg in feed

Controls 125 20 113(3-512) 50(2-287)* -55.8

Control animals were unavailable after December 2, 1959.

Table 13. Analyses of variance In EPG counts of Crofoot Feedlot test.

:

Source of Variation * d.f. Ms F • • •

Treatment 2 139.990 10.69 *** Animal s:Trts. 57 18.569 1.42 *** Periods 3 294.7197 22.50 *** Periods x Trts. 6 20.8014 1.59 ns Periods x Animals :Trts. 170 13.0971 m •

*** P < .001 J

en c — c > c — • « — I «° Ui

(0 (0 s B £| —

— +j 3 XI O o c o s Q. X — CO t> 1**~- X •M 4) X> O x c 36

< -3

I I

Ul UJ > > -j a LJ UJ o UJ _l _1 IX i- X H < o * Z UJ o o ac I -i o t-

CO HUJ < UJ Q O Ol Z -J a. 2 < <0

o o

9d3 IVIOI 37

drench which indicates that Ruelene is more adverse in its effects* both against the parasites and the host (Table 15). However, opposite results have been observed in other tests comparing the same drugs (Norris, 1961; personal communication). Because there were no data available on the indi- vidual weights of each animal no statistical analyses could be performed.

Analyses of variance of the raw EPG data showed that Ruelene treatment resulted in a higher percentage reduction of egg counts (Table 16). Again, grouping the EPG data into groups (0-299 and 300 up) increased the signifi- cance of the differences (Table 17).

Ruelene Oral Versus Dermal Test (Table 18-21). Calves of the same type and quality as those used in the Cleburne Feedlot Test were used to compare feed and pour-on treatments of Ruelene relative to EPG counts.

Eight of 100 head In one pen were tagged in the right ear and fecal sam- ples were taken. The animals in this lot had been taken off feed 12 hours earlier. Ruelene formulation M-1839. using 6.) lbs., was mtxed with the total ration of 3500 lbs. to provide a dosage of k5 mg/kg/head. In another pen of

100 head, 2k were tagged and fecal samples taken. Ruelene formulation M-1609 was administered as a pour-on at the rate of 75 mg/kg to all except 12 of the tagged animals which were controls. Post-treatment fecal samples were again taken from each group 15 days later (Table 18).

Statistical analyses of the EPG data showed that method of administration of Ruelene had no significant effect on the egg count results (Table 19)*

However, the results approached significance after the EPG data were grouped

(Table 20). The difference between the calculated F value of the data and

the F value at the .05 level is less in the grouped data than in the non- grouped (Table 21). 38

Table 14, Experimental design of the Ruelene vs Phenothiazine-Lead Arsenate Test.

. _ fc „ . No. . No. . Average EPG Count & Range Per Cent

. 11/1/60 Reduction ; Trtd. : Smpld. : 11/16/60 :

Ruelene 32 9 368(39-851) 3(0-11) -99.2 75 mg/kg drench

Pheno- 36 10 251(13-842) 86(3-442) -61.8 thlazine- Pb (As0 3 4 ) 2 drench

Table 15. Ruelene versus Phenothiazine-Lead Arsenate Test.

Pre-treatment : Post-treatment Average

Treatment ', 11/1/60 ; 11/16/60 Gain (its.) : Avg. Wt. : No. : Avg. Wt. : No. ;

Ruelene 329.37 32 349.31 29 19.94

Pheno- 325.28 36 356.0 10 30.72 thlazine*

*Phenothlazine, 15 gm and Lead Arsenate, 0.25 gm.

Table 16. Analyses of variance In EPG counts of Ruelene and Phenothtazine treatments.

Source of Variation d.f. * Ms F • • •

Treatments 1 406725.06 7.79* Animal :Trts. 14 52241.21

*P < .05

Table 17. Analyses of variance of EPG data of Ruelene and Phenothiazlne treated calves after grouping.

Source of Variation d.f. ' Ms F

Treatments 1 406725. 06 16.34*** Animals:Trts:Grps. 12 24886.56

*** P < .001 39

Table 18. Ruelene Oral versus Dermal Test.

EPG Count & Range Per Cent . No. . No. . Average Treatment • : : Reduction : Trtd. : Smpld, : 10/31/60 11/15/60

Ruelene 100 8 126(3-331) 13(0-52) -89.7 45 mg/kg In feed

Ruelene 88 12 206(6-801) 26(1-83) -88 .4 75 mg/kg topical

Controls 12 12 353(8-2324) 240(3-1784) -32.0

Table 19. Statistical analyses of EPG data from the Ruelene Oral versus Dermal Test.

Source of Variation d.f. Ms

Treatments 2 14350.40 .41 ns Animals:Trts 27 34737.61

Table 20. Statistical analyses of the grouped EPG data of the Ruelene Oral versus Dermal Test.

Source of Variation d.f. Ms

Treatments 2 11*350.40 2.88 ns Animals:Trts:Grps. 19 4979.75

Table 21. Effect of grouping EPG counts on the F. value.

Calculated EPG Data F at .05 Difference F

Non-grouped .41 3.35 2.94 Grouped 2.88 3.52 .64 40

Nacogdoches Bull and Heifer Test . Bulls and heifers, weighing an average of 650 and 500 lbs., respectively, were separated Into two lots by sex. The animals were numbered and rectal fecal samples were taken before and two weeks fter treatment.

Ruelene was administered In varying doses as a pour-on and drench to all animals of both sexes except those designated as controls (Tables 22 and

23).

Although post-treatment EPG counts were reduced substantially, analyses of variance showed that the differences were not significant (Table 2k), The data were not grouped because of the small number of animals sampled from each treatment.

Larval Identification

In order to Identify the larval stages of cattle nematodes, It Is advan- tageous that they be relaxed (straightened) for measuring. Because It was

Inconvenient to identify the larvae upon arrival from the Texas Phenothiazlne

Company Laboratory In Texas, It was necessary to develop a method to simul- taneously straighten and preserve them. Hot 70 per cent alcohol or hot forma- lin proved unsatisfactory because many of the larvae were not straightened.

Shivnanl and Hansen (1956) applied heat directly to the slide In order to straighten larvae. Because the larvae were to be preserved for later identi- fication, heat was applied by immersing the vials, containing the larvae, Into boiling water. Optimum time that the larvae were to be heated was determined by a preliminary experiment using Infective larvae of Trichostrongylus axel

and T. colubriformis . Twenty seconds proved optimal when using cold fixative

(Table 25). Keith (1953) used heat sparingly to avoid shrtnkage of the larvae 41

Table 22. EPG counts of Bull Test.

—, .I

.' * No. No. ! Average EPG Count & Range Per Cent Treatment j Trtd. Smpld. 10/29/60 . 11/12/60 j : : : Change

Rue 1 ene 5 4 417(357-486) 93(21-239) -77,7 drench

Ruelene 22 12 205(9-1231) 93(9-613) -54.7 pour-on

Controls 6 3 352(50-930) 437(42-740) +27.8

Table 23. EPG counts of Heifer Test.

- — : No. : No. Average EPG Count & Range : Per Cent :

: Trtd. : Smpld. : 10/29/60 : 11/12/60 : Change

Ruelene 7 6 176(1-430) 20(0-39) -88.7 drench

Ruelene 7 3 178(17-365) 66(37-8^) -63.0 pour-on

Controls 7 6 136(2-589) 141(2-487) + 3.6

Table 24. Analyses of variance of the combined EPG data of the Bull and Heifer Test.

Source of Variation d.f. Ms

Treatments 5 90399.59 2.06 ns Anima)s:Trts. 28 ^3962.87 kZ

m •u §§> O NO o o o o • u — • •III I I I I •••• TO 4- k »A CO o o o 0) 4J a. *a jj 1 • • • » o -J i— 5 | TO IA IA o q no L> TO CM CM ft > O • I • k 1- TO • • -J *j

«*- en NO I O I IA NO TO co 8 3 • k o 4-> >ui z V> • «» o 1- to < > M JJ 4J k C £ TO H — • • • • • • • TO CAI-*QO«MIOO — nOI I I I c U k co booojt00 O Jf o o o\ o> o O *J a. v> e > • • •• U | »» (0 fc X 9 i TO CO .tflAOCM 00 IA NO N0 t — r TO +J _ , _ _ _ j- i — m cr» i i i t 14- > O o — o k 1- •o TO • • «J 4-1 8 lb * 1. *TO S.^^SS.^^gcA • k •A 0\ •u w z V) X o § % ^-» u TO 3 c Nor^roMr*.r^NOiACMONO"-o>iJfo j M 1 — J-J-iAnOnOJ"^iAnOIAIA NO NO f» CO w TO lA. X k en I c a •» • • «• 4J Hty s ,__ X • (0 MM c TO <+- ft tm C en a^ss^assacA ,a a ca a a § •M «/> c o • (A a (A TO CJ . kO UkCIO en 0> 33 en c TO -W4J c ._ a) Q TO TO •*• +j e kk TO — lAIAOIAOIAIAOIAIAOlAqqO I « t- — — CM 5 «• — — CM CO J- #* X o tc IB

• s si to a> u o caoc — o — N m 4 « TO z H 43

within the sheath. Although some shrinkage was noted In the preserved samples,

Oesophagostomum It did not hinder identification. The third stage larvae of

identified. were the roost affected by heat. However, It could be easily

The method of handling the larvae was as follows. After the samples had

attained room temperature and the larvae had settled to the bottom of the vial

milliliters the supernatant was removed with the aid of an eye dropper. Two

vial. The of solution, containing the larvae, were left In the bottom of the

supernatant was examined under a dissecting microscope to make sure no larvae

had been removed. Fifteen vials were placed In a wire basket constructed to

beaker of boil- fit Inside an 800 ml. beaker. The basket was lowered Into the

from the boil- ing water for 20 seconds and agitated carefully. After removal

per cent ing water, the contents of each vial were doubled by adding cold 20

formalin. This resulted In a 10 per cent formalin solution to preserve the

larvae until It was convenient to Identify and count them.

The vials containing the preserved larvae were shaken and examined under

of larvae. Larvae a dissecting scope to determine the relative concentration

were concentrated, when necessary, by decantatlon of the supernatant. The

supernatant was examined In a watch glass under a dissecting scope and any

larvae found were returned to the proper vial.

the Each vial was agitated by hand for one minute, then three drops of

contents were quickly removed with an eye dropper. The drops were placed In

the middle of a 2x3 Inch glass slide that had previously been prepared by

scratching 2 lines through the center at right angles to each other. A cover

glass (18x18 mm. No. 2) was held directly over the center and carefully

dropped on the suspension of larvae. Using the vertical line for a reference

cover slip. mark, all larvae up to 25 were counted under the right half of the I*

If 25 were not present under the first half, the larvae on the left side of the reference mark were counted. To avoid counting any larva more than once, each was counted In the field showing the posterior end. All slide samples were carefully examined using the same technique. This procedure was repeated k times until 100 larvae were counted from each vial. After every 25 larvae counted, the slide was washed In 70 per cent alcohol and wiped dry.

In some cases the number of larvae present was so small that the sample could not be concentrated enough to provide 25 larvae per 3 drops. When this occurred, the entire sample was examined In a watch glass under a dissecting scope. All larvae were picked up with a small pipette and placed on the slide for Identification.

The larvae were Identified and counted simultaneously. The number of each species per 25 counted was recorded on an Adams Laboratory Counter. After every 25, the totals were recorded. Identification of the larvae was done according to Hansen and Shlvnanl (1956) and with the aid of the nomogram by

Oewhlrst and Hansen (1961). Identification of the larvae served a two-fold purpose in this study, the first being the determination of the relative sus- ceptibility of the various species to Ruelene or phenothlazlne treatments, and the second to determine the degree of accuracy of the egg counts.

The larvae of Strongyloses paplllosus were easily Identified by their

long esophagus. However, because the eggs of this species were not counted, neither were the larvae.

The pre-treatment larval samples from Ruelene treated animals were compared with the post-treatment larval counts of the same animals (Table 26).

The total larva' counts are the sum of five Ruelene treatments from three

experiments. The differences between the pre- and post-treatment larval kS

counts of the Ruelene regimens were highly significant (P < .001). Comparison of the larval counts from the control animals of the same experiments revealed

no significant changes in the number of each species of larvae (Table 27).

Therefore, Ruelene showed activity against certain nematodes present In the

test animals.

Ruelene appeared to be most effective against Haemonchus and Cooper I a

and least active against Trichostrongylus and Ostertagla (Table 28).

Phenothlazine was used in only one experiment. Again, comparisons of the

pre- and post-treatment larval counts of the phenothlazine treated animals

showed that certain nematodes were more susceptible than others (Table 29).

Chl-square showed the differences to be highly significant (P < .001), Indi-

cating that nematocldal activity of the drug varied among the parasites pres-

ent. The results of the phenothlazine larval counts are similar to those

obtained with Ruelene as the HO group appeared more susceptible, while

Ostertagla was the least affected (Table 30).

To determine the accuracy of the differential egg count method employed

In this study, the percentages of larvae In the Bunostomum . COT and HO groups

were compared with the percentages of eggs for corresponding groups. Only

samples that had both EPG and larval counts were Included in the percentage

calculations. Table 31 compares the larval and EPG counts of cattle used in

the Ruelene experiments. The comparisons on the animals used in the pheno-

thlazine experiment were tabulated separately (Table 32). It must be noted

that only certain species of larvae can be Identified using the method of

Oewhlrst and Hansen (1961). Nematodlrus was not Included in the larval counts

because the cul'.ure period was too short for its development. Therefore,

the eggs of Nematodlrus were Included with those of Trlchurls and Capillarla . 46

before and after Table 26. Comparison of the numbers of each larval species Treatment with Ruelene.

Species . _, ; t^otal* c unct - 1 Ostert. Sample :Buno.: -P ^Concho. 1 Haem. :0esoph. Trlcho.: .C.pect. : : : : : : f

Pre 120 2364 340 892 185 104 579 4584

Post 64 929 43 17 3 250 670 1976

* Chl-square highly significant (P < .001).

the control Table 27. Comparison of the numbers of each larval species in groups.

Species : Tota C.puntt. : * Sample :Buno #: :c.oncho. 1 Haem. :Qesoph. : Trlcho.: Ostert. . : : : C.pect. : : :

Pre 70 1114 106 336 39 39 282 1986

Post 48 657 58 220 21 30 182 1216

* Chl-square not significant.

treatments Table 28. Susceptibility of some nematodes of cattle to Ruelene determined by larval counts.

• Per Cent of Each Species t Net Difference rarasUe KanK . , p re- trt# Post-trt. : (%)

1 Haemonchu* 19.4 0.8 -18.6 2 Cooperia onchophora 7.4 2.1 - 5.3 3 C. punctata and 51.5 47.0 - 4.5 C. pectinata k Oesophagostomum 4.0 0.1 - 3.9 5 Bunostomum 2.6 3.2 + 0.6 6 Trlchostrongylus 2.2 12.6 +10.4 * 7 Ostertagia 12.9 34.2 +22.3 k7

Table 29. Comparison of pre- and post-treatment larval counts of phenothfazine treated animals.

• Spec! es

Sample: Buno# :C.punct. : Concho. : Haem. : Oesoph. : Trlcho. '•Ostert. jTotal* • • • • • : rC.pect. • • • • •

Pre 52 *M7 19 168 8 5 68 737

Post 62 208 1 21 71 363

* Chl-square highly significant (P < .001).

Table 30. Susceptibility of some parasites of cattle to phenothiazine determined by larval counts.

of Each Species . Met Difference : Per Cent Rank t Parastte - Pre-trt. Post-trt. : : :

-22.5 1 Haemonchus 22.7 0.2 - 2 Cooper i a onchophora 2.5 0.0 2.5 - 1.0 3 Oesophagostomum 1.0 0.0 k C. punctata and 56.5 57.3 • 0.8 C. pectinata 5 Trichostrongylus 0.6 5.7 + 5.1 6 Bunostomum 7.0 17.0 +10.0 + 7 Ostertagia 9.7 19.8 10.1 w

The eggs of these genera constituted a group (All Other), which In all cases,

was of no consequence In the animals studied.

Error of classification of eggs Into COT or HO groups Is evidenced by the

disagreement between larvae and EPG counts of Groups I, II and IV, given In

Table 31. For example, the percentage of larvae and eggs for the COT species,

Group I, Table 31, was 72.7 and 91.8, respectively, a difference of 19.1 per

cent. The difference between the larval and EPG counts of both the COT and HO

species In the Ruelene experiments ranged from 17.3"21.2 per cent, excluding

Group III. The other groups could be combined to determine the error because

Group III was the only one affected by treatment. The mean difference is 18.75

per cent, which Is also the average error. Assuming that the larval percent-

ages were accurate, then 18.75 per cent of the eggs classified as COT should

have been classified as HO species. Subtraction of this amount from the COT

EPG percentage and addition of the same to the corresponding HO percentage

reconciles the differences of Groups I, II and IV of the Ruelene experiments.

Using the same calculation procedure, the error for Group III was 0.85 per

cent. In the phenothlazlne experiment the error was 22.5 and k.2 per cent

before and after treatment, respectively.

DISCUSSION

The evaluation of an anthelmintic Is based upon Its effect on fecal egg

counts supported by critical examination of the Intestinal tract. When using

egg counts for evaluation of an anthelmintic, technician errors are considered

to be consistent throughout the experiment. The activity of a compound Is

often measured by computing the per cent reduction In the post-treatment egg

count. Using the egg reduction criterion only, It Is Impossible to dlstln- 49

Table 31. Comparison of the larval and EPG diagnostic techniques used In Ruelene experiments.

Species . Diagnostic (%) Group . '. Technique COT : HO : Buno. : All Other

1. (Pre-treatment Larvae 72.7 24.3 2.5 0.0 Test Animals) EPG 91.8 7.* 0.6 0.2

II. (Pre-treatment Larvae 75.6 21.0 3.4 0.0 Controls) EPG 95.62 3.7 0.6 0.08

III. (Post- treatment Larvae 96.5 1.3 2.2 0.0 Test Animals) EPG 97.8 0.9 0.8 0.5

IV. (Post-treatment Larvae 74.9 21.0 4.1 0.0 Controls) EPG 96.1 2.5 1.2 0.2

Table 32. Comparison of the larval and EPG diagnostic techniques used In the phenothiazlne experiment.

Diagnostic Species (%) Group ; : Buno. All Other : Technique { COT : HO : :

1. (Pre-treatment) Larvae 69.2 23.8 7.0 0.0 EPG 91.4 1.0 7.3 0.3

II. (Post-treatment) Larvae 30.0 0.4 19.6 0.0 EPG 88.1 0.1 10.3 1.5 50

guish between actual worm removal and ovulation suppression without the support of critical test data. In the present study the animals were fed from bunkers, thus almost eliminating the chance of reinfection, which justifies the single post-treatment sampling technique. The type of critical testing used in this study did not detect prepatent larvae because they can only be recovered by artificial digestion of organs. Therefore, evaluation of the compounds was based on activity against adjlt nematodes.

This study revealed that the statistical method employed is important when analyzing EPG data. The analyses of variance of raw data In the study showed that the treatments had no valid effect on the egg counts even though critical tests showed pronounced anthelmintic activity. Values of egg counts had an inherent large variation within test groups which unduly influenced the statistical results between groups. When egg counts were separated into groups of a smaller specific range (0-99; 100-199, etc.), the statistical results were consistent with the results obtained by critical tests (Wood,

1961; personal communication).

Grouping egg counts can be a valuable aid to anthelmintic studies con- ducted under usual field conditions. In such studies cattle of different breeds, conditions, worm burdens and ages would be encountered. Of course, from the experimental standpoint, It is better to have the treated animals similar to the con.'ols. This would involve setting up the lots of cattle on the basis of pre-treatment egg counts, which Is impossible in many cases.

Both the treated and control animals should have a similar average and range of egg counts.

The accuracy of the differential egg count method used In this study was determined by comparison of EPG and larval counts. The total counts 51

were converted to percentages for each species or group because the larva)

counts were not quantitative as were the egg counts. The error in classify-

ing eggs for the phenothiazlne and Ruelene experiments was calculated sep-

arately In order to detect any selective action of these drugs against

different genera of nematodes. However, the results were the same in both

cases. Before treatment there was a considerable error (18.75*22.5%) In both

phenothiazlne and Ruelene experiments while after treatment the error was markedly (0.85-4.2%) reduced. The increased accuracy in egg classification

was related to the elimination of nematodes whose eggs were difficult to

classify. This conclusion is supported by two facts. First, results of

larval studies showed that the HO group was more susceptible to the anthel-

mintics used than the COT group. Secondly, the eggs of the HO group are

definitely more difficult to classify. Eggs of Haernonchus can easily be

mistaken as ova of Ostertagia (Norris, I960; personal communication).

The results of the egg and larval count comparisons emphasize the fact

that larvae are more accurately Identified than eggs. However, in most cases

the convenience of the less accurate egg count method Is preferred to the time

consuming larval culturing technique. If the person performing the egg counts

is experienced, the error will be at a minimum. The author had performed over

300 egg counts on bovine fecal samples prior to those included in the accuracy

determinations. It Is difficult to set a definite number of egg counts as the point whereby the technician would be considered experienced. However, In

view of the error obtained In this study, 300 should be below the minimum

number, if a standard were established.

In order for the novice to gain experience quickly, larval and egg counts

should be performed concurrently. Training the technician by this procedure a

52

Mould Increase the accuracy and efficiency of the technique. Establishment of a standard for experienced technicians could then be on the basts of attaining a certain amount of accuracy. Although, In either case, experience is the roost vital part In performing an accurate differential fecal egg count.

In all of the conducted tests, Ruelene proved effective against

of Haemonchus and Cooper i In beef cattle. Comparing the 3 methods adminis- tration, the pour-on technique, with an overall average EPG reduction of 72.4 per cent, was the least effective. Ruelene fed with the ration was the roost effective with an average reduction of 90.4 per cent. The average reduction obtained with Ruelene as a drench was 80.9 per cent, although drenching was the least consistent as the reductions varied over a wider range (58.0-99.2 per cent).

A possible explanation for the greater variation obtained when using a drench is the effect produced by the esophageal groove. Several workers have noted a difference in the efficacy of an anthelmintic depending upon whether

It was Introduced Into the rumen or the abomasum. Gordon (1958a), Riek

(1958), Rlek and Keith (1958) and Ounsmore (i960) all found the anthelmintics

they were testing to be more effective when directed into the abomasum. They stimulated closure of the groove by swabbing the pharynx with a 10 per cent

CuSO^ solution. According to Dukes (1955) the reflex Is evoked immediately after the stimulant comes Into contact with the pharyngeal mucous and will

last for 15 seconds or longer. Thus, closing the groove and directing the

drug Into the abomasum results In greater concentration of the compound. If

the anthelmintic Is swallowed Into the rumen, action against the gastroin-

testinal parasites probably comes through the circulatory system, In the case of systemlcs, as they feed upon the blood and tissue fluids. Via this route, 53

concentration of the compound is undoubtedly less than if the drug were

introduced directly into the abomasum.

The results of Herlich and Porter (1958) on the efficacy of Bayer 21/199 led them to believe that possibly the anthelmintic action was directly against the worms rather than via the circulatory system. This could possibly be due to a maximum concentration of the drug In the abomasum. Although they did not purposely stimulate the esophageal groove reflex, It may have occurred natur- ally according to Gordon (1958b). Even though introduction of the nematocide into the abomasum to obtain maximum concentration indicates the possible direct action against the parasites, another alternative should be considered.

That Is the possibility of the nematodes Ingesting greater quantities of the drug from the circulatory system or tissues due to the high concentration of the compound taken directly into the abomasal lining.

Resolvement of the possibilities depends upon the determination of the actual mode of action of anthelmintics of the organic phosphorous type, of which at the present time, much Is yet to be learned.

SUMMARY

Studies were conducted on 1,115 beef cattle to determine the efficacy of an organic phosphorous systemic insecticide, Ruelene, against gastrointestinal nematodiasis. The drug was tested at different levels, administered as a drench, pour-on or feed additive. One comparative study included the efficacy of Ruelene versus phenothiazlne'lead arsenate.

Ruelene, fed with the ration over a five day period, significantly reduced

EPG's by 37.8 and 32.2 per cent more than those of the controls as a high or low level (50 and 15 mg/kg) regimen, respectively. 54

In one test, Ruelene, as a 45 rag/kg dose fed with the ration, was compared with a pour-on at a 75 mg/kg rate. The EPG reductions were non-significant, although they were 57.7 and 56.4 per cent lower than the controls, respectively.

Bulls and heifers, divided into lots by sex, were used to compare the effects of Ruelene administered as a drench or pour-on. The drench was more effective as it reduced EPG's by 23.0 and 25.7 per cent more than the pour-on

In both the bull and heifer tests, respectively.

In direct comparison, Ruelene, given with the feed at the rate of 45 mg/kg, reduced EPG counts by 8S.7 per cent while a 75 mg/kg drench reduced egg counts by 88.4 per cent. Controls were down 32.0 per cent.

Administration of a Ruelene drench at the rate of 75 mg/kg reduced egg counts by 99.2 per cent as compared to the 61.8 per cent reduction of a phenothlazlne-lead arsenate drench.

In one trial, Ruelene, administered as a 50 mg/kg drench and as a pour-on at 2 rates, 50 and 75 mg/kg, reduced egg counts by 58.0, 84.3 and 73.7 per cent, respectively. The egg counts of the controls were down 19. 9 per cent.

Comparison of the critical test data of five animals showed that Ruelene

was highly effective against Haemonchus and Cooper I a . tittle or no effect was evidenced against the other species present.

A method for the mass preservation of straightened infective larvae was devised utilizing boiling water as a killing and straightening agent.

Identification of infective larvae was used to determine the more suscep- tible species of nematodes to the anthelmintic action of Ruelene or phenothi- azlne. Haemonchus and Cooper la spp. were the most affected by Ruelene while

Trlchostrongylus and Ostertagia were the least susceptible. Phenothlazlne

produced similar results as Haemonchus , JC. onchophora and Oesophagostomum 55

were the most susceptible, while Bunostomum and Ostertaqla were the least affected.

Larval Identification data were also used to test the accuracy of the differential egg count method. The error In classification of eggs prior to treatment was 18.75 and 22.5 per cent in the Ruelene and phenothiazlne tests, respectively. After treatment the error was 0.85 and k.2 per cent for the same respective drugs. This reduction in error of classification was related to the elimination of nematodes whose eggs were difficult to classify. ACKNOWLEDGMENTS

For the advice and aid provided by Or. Merle F. Hansen, major advisor,

during the course of this study, my slncerest appreciation Is expressed.

A special thanks Is extended to Mr. Mark G. Norris, Jr., of Dow Chemical

Company, for the technical assistance and materials he provided. To Or. Fred

W. Knapp, University of Kentucky, for the initial guidance provided, my

sincere thanks is expressed. Indebtedness is also acknowledged to the members

of the Zoology Department, who aided In the accomplishment of this study.

Indebtedness Is acknowledged to Crofoot and Son of Strong City, Kansas, for

their cooperation and facilities which were utilized in this study.

To Or. Stanley Wearden of the Statistical Laboratory at Kansas State

University, my gratitude Is expressed.

For the financial support of this study, acknowledgment Is made to the

Agricultural Experiment Station, who provided my asslstantshlp.

The author is also grateful to his wife, Eleanor, for the patient,

understanding encouragement given during the course of this study. 57

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Worley, D. E. Ecological and therapeutic studies on gastrointestinal parasitism in beef cattle in Kansas. Unpublished Ph.D. thesis, Kansas State University. 1958. THE EFFICACY OF RUELENE AS AN ANTHELMINTIC IN BEEF CATTLE

by

DANIEL ALBERT OSTLINO

B. S., Bethany College, 1958

AN ABSTRACT OF A THESIS

submitted In partial fulfillment of the

requirements for the degree

MASTER OF SCIENCE

Parasitology

Department of Zoology

KANSAS STATE UNIVERSITY Manhattan, Kansas

1962 A study utilizing 1,115 beef cattle was conducted to investigate the

anthelmintic activity of Ruelene, an organic phosphorus insecticide, and phenothiazine. The experimental design used 193 animals for egg counts

and 6 animals for critical tests.

The anthelmintic Investigations involved field trials of the drug, varying both the dosage and method of administration. The methods utilized

included drenching, pour-on (a new topical application) and feed addition.

Ruelene was given at the rates of 50 or 75 mg/kg as a drench or pour-on.

When fed as a premix in the ration the rates were 15, k5 and 50 mg/kg in various tests. In most of the tests egg counts were made on fecal samples taken directly from the rectum. In each method of application Ruelene significantly reduced EPG counts. Orenching produced better results than the pour-on, although it was not as consistent. Ruelene produced the highest average egg count reduction when given in the feed. The topical application was the least effective. In a single comparative test, Ruelene as a drench reduced egg counts more than a phenothiazine- lead arsenate drench.

Critical examination of the intestinal tracts of six animals demonstrated the effective anthelmintic activity of Ruelene, particularly against

Haemonchus and Cooper la .

Larvae were cultured and identified to determine the nematocidal affect against various species harbored by the cattle. Haemonchus spp. and Cooper la spp. were the most susceptible to treatment while Trichostrongylus spp. and

Ostertagia spp. were more resistant.

The larval identification data were compared to the corresponding EPG data to determine the accuracy of the differential egg count technique.

Approximately 19 per cent of the ova counted were mtsclassif ied.